biostudies-arrayexpressCarlos TorrojaSus scrofahttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14275Most of the mechanistic insights into atrial fibrillation (AF) pathophysiology have been reported on cardiomyocytes, and it is commonly assumed that such apply in the same manner to the atria as a whole. This study shows that atrial remodeling during persistent AF (PsAF) underlies differential adaptative changes in non-myocyte populations, which depend on the functional relevance of individual-specific atrial regions to drive the overall arrhythmia. Radiofrequency delivery at driver regions can terminate the arrhythmia in pigs and achieve long-term arrhythmia AF-free survival in patients. Importantly, driver regions show overt compositional shifts in fibroblasts and myeloid populations that are highly consistent across pig models and patients. More specifically, driver regions underlie a phenotypic shift towards cardiac resident macrophages with an associated transcriptomic and proteomic profile favoring cardiomyocyte homeostasis and cell survival within a substrate prone to reentry. In fibroblasts, PTX3 represented a transcriptional hallmark exclusively present in driver regions, which supports their role on modulating the immune response of the surrounding tissue.biostudies-arrayexpressSample Collection - Hearts were harvested and dissected, single cell suspension was prepared by enzymatic digestion. Tissue was minced to ~1 mm3 cubes and digested in 3 mL of DMEM containing 450 U/mL collagenase I, 60 U/mL DNase I, and 60 U/mL hyaluronidase (all Sigma- Aldrich, Munich, Germany) for 60 min at 37º C. The resulting cell suspension was filtered through a 70-μm nylon mesh (Corning, USA) to remove undigested tissue fragments, and pelleted by centrifugation (400 × g for 5 min at 4ºC). Cells were re-suspended in fluorescence activated cell sorting (FACS) buffer (phosphate-buffered saline [PBS] with 1% foetal calf serum + 0.1% bovine serum albumin) before antibody labeling and FACS sorting. For preparation of porcine cell samples for scRNA-seq analysis, viable and nucleated (Sytox Blue- DRAQ5+) endothelial (CD31+ CD45-), leukocyte (CD45+) and other non myocytes (CD31- CD45-) were sorted in FACS Aria II cell sorter (BD biosenses). Then, cells were resuspended in PBS supplemented with 0.04% bovine serum albumin. Cell viability was then checked using an automated Countess III cell counter with software v1.0.296.782 (Thermo Fisher Scientific) in all single cell suspension composed of approximately 20% endothelial cells, 70% leukocytes and 10% other non-myocytes.Sample Treatment - Yucatan-Large White crossbred pigs were used to generate the long-lasting lone AF model (N=19) and sham-operated controls (N=8). A dual chamber pacemaker (Accent DR-RF, St. Jude Medical, CA, USA) was surgically implanted in the subcutaneous tissue of the right side of the neck. Atrial and ventricular leads were (Tendril STS, Abbott, MN, USA) inserted through the jugular vein and then positioned in the right atrial appendage and right ventricular apex or septum using active fixation electrodes. Ten days after the pacemaker implantation and animal recovery, a second procedure was performed to ablate de atrio-ventricular node. The latter aimed to prevent rapid atrio-ventricular node conduction and heart failure upon starting the AF induction protocol. The ablation procedure was performed using an 8-mm tip ablation catheter (Blazer II XP, Boston Scientific Corporation, Marlborough, USA), which was introduced through an 8-French introducer (ENGAGE Introducer Sheaths, Abbot, MN, USA) in the right femoral vein. Then, the ablation catheter was positioned at the atrio-ventricular node under fluoroscopic guidance and radiofrequency energy was delivered using a temperature control mode with power set at 70W and a cut-off temperature of 70ºC. Complete atrio-ventricular block was documented using intracardiac electrograms and surface ECG recordings. Then, the atrial pacing protocol was activated to induce AF. The atrial pacing protocol consisted of a 30-second burst pacing at 20 Hz and twice diastolic threshold, followed by a 6-second sensing period. If the pacemaker detected sinus rhythm restoration during the sensing period, high-rate atrial pacing was automatically reinitiated to induce a new AF episode. Atrial electrograms during AF episodes were recorded and stored using the automatic switch-mode algorithm of the pacemaker device. The same procedures were performed in sham-operated controls, but the high-rate atrial pacing protocol was not activated and the pacemaker was programmed in DDDR mode after atrio-ventricular node ablation. The procedures were performed after pre-medication (intramuscular ketamine 20 mg/kg and midazolam 0.5 mg/kg), anesthesia induction (intravenous fentanyl 0.010 mg/kg). After endotracheal intubation, pigs were mechanically ventilated using a volumetric ventilator (tidal volume: 10 ml/kg, respiratory rate: 14 breaths per minute, maximum inspiratory pressure: 25 mm H2O, intermittent positive end expiratory pressure: 4 mm H2O). Anesthesia was maintained using 2% sevoflurane. Electrocardiogram, oxygen and carbon dioxide levels were monitored throughout the procedure. Some pigs underwent also a procedure of ischemia-reperfusion (3-hour ischemia followed by 1 hour or continuous monitoring during reperfusion) at the proximal left circumflex artery. The procedure follows a similar approach to previous reports of ischemia-reperfusion to generate infarct-related substrates.39 Briefly, the procedure was performed using percutaneous access thorough an 8-French introducer (ENGAGE Introducer Sheaths, Abbot) in the right femoral artery. A bolus of intravenous heparin was administered (300 IU/kg) at the beginning of the procedure. Then, a 5-French AL-2 guide catheter (Cordis, FL, USA) was progressed until the left coronary trunk to assess the coronary anatomy with two angiographic projections. Then, the catheter tip was positioned into the proximal circumflex artery with the use of a 0.014-inch guidewire. Thereafter, an angioplasty balloon (3.5-mm diameter balloon, Medtronic, MN, USA) was positioned into the proximal circumflex artery and further inflated 462 to generate ischemia in both the lateral-basal part of the left ventricle and the left atrium. Appropriate balloon inflation and artery occlusion were confirmed on the coronary angiogram. The artery was occluded for 3 hours before reperfusion. Intravenous amiodarone (300 mg/h) was administered throughout the procedure to decrease the risk of ventricular arrhythmia. The procedure was performed using the same pre-medication and anesthesia protocol described for pacemaker implantation and atrio ventricular node ablation. Eight weeks after ischemia-reperfusion in the circumflex artery the animals underwent the same protocol described for the lone persistent AF model.Nucleic Acid Extraction - Single-cell suspensions were loaded into a Chromium Next GEM Chip G (10x Genomics) to capture a maximum of 10,000 single cells per port utilizing the Chromium Controller (10x Genomics).Sequencing - Library pool was sequenced using one P3 flow cell (100 cycles) on a NextSeq 2000 (Illumina). FastQ files for each sample were obtained using cellranger mkfastq pipeline (10x Genomics).Library Construction - scRNA-seq libraries were prepared using the Chromium Next GEM Single-Cell 3′ Kit v3.1 (10x Genomics). Individual libraries were diluted to a concentration of 10 nM and pooled for sequencing.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - CellRanger v6.1.1. pipeline was used to generate a digital gene expression matrix starting from raw data. For alignment and quantification of gene expression, the reference transcriptome was built using genome pig Ss11.1 with ensembl gene build version 109 (feb2023.archive.ensembl.org).Data Transformation - The raw digital gene expression matrix (UMI counts per gene per cell) was imported in R https://www.R-project.org/ version 3.5.0 and processed using scater for quality control and filtering purposes and Seurat R package (version Seurat_2.3.3) for clustering and subsequent analysis. We obtained data from 91,774 cells and 31,781 genes that passed quality control steps implemented in Cell Ranger. Then, we filtered out cells meeting any of the following criteria: 400 unique genes expressed, <1,000 or >30,000 unique molecular identifiers, >25% of reads mapping to mitochondria, <0.2% cell counts fraction in the sample, cells with high levels of reads in just a few genes, >65% of reads in the top 50 genes and > 0.1% of reads in hemoglobin subunits (Extended Data Fig. 12). Doublets were detected and filtered out using scDblFinder4 with default settings by sample. Filtered cells were then log-normalized and a total of 1,000 most variable genes (vst method) were selected for subsequent dimensionality reduction and clustering steps. After principal component analysis dimensionality reduction, a nearest-neighbor graph followed by Louvain clustering was applied using the first 20 components of the principal component analysis. Uniform Manifold Approximation and Projection Plot (UMAP) dimensionality reduction on the same 20 components was applied for visualization.UnknownTranscriptomicsGenomicsProteomicsNextSeq 2000RNA-seq of coding RNA from single cellsSus scrofaCarlos TorrojaDavid Filgueiras-RamaAna Simon-ChicafalseRegional specific changes in cardiac resident 1 macrophages and 2 fibroblasts modulate long-term atrial fibrillation maintenanceMost of the mechanistic insights into atrial fibrillation (AF) pathophysiology have been reported on cardiomyocytes, and it is commonly assumed that such apply in the same manner to the atria as a whole. This study shows that atrial remodeling during persistent AF (PsAF) underlies differential adaptative changes in non-myocyte populations, which depend on the functional relevance of individual-specific atrial regions to drive the overall arrhythmia. Radiofrequency delivery at driver regions can terminate the arrhythmia in pigs and achieve long-term arrhythmia AF-free survival in patients. Importantly, driver regions show overt compositional shifts in fibroblasts and myeloid populations that are highly consistent across pig models and patients. More specifically, driver regions underlie a phenotypic shift towards cardiac resident macrophages with an associated transcriptomic and proteomic profile favoring cardiomyocyte homeostasis and cell survival within a substrate prone to reentry. In fibroblasts, PTX3 represented a transcriptional hallmark exclusively present in driver regions, which supports their role on modulating the immune response of the surrounding tissue.2025-11-06T00:00:00Z2025-11-06T18:12:49.713Z2024-07-22T12:24:51.313ZE-MTAB-14275ERP162205EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0003969EFO_0004184